Identifying Early Infections in the Setting of CRS With Routine and Exploratory Serum Proteomics and the HT10 Score Following CD19 CAR-T for Relapsed/Refractory B-NHL.

Early fever after chimeric antigen receptor T-cell (CAR-T) therapy can reflect both an infection or cytokine release syndrome (CRS). Identifying early infections in the setting of CRS and neutropenia represents an unresolved clinical challenge. In this retrospective observational analysis, early fever events (day 0-30) were characterized as infection versus CRS in 62 patients treated with standard-of-care CD19.CAR-T for relapsed/refractory B-cell non-Hodgkin lymphoma. Routine serum inflammatory markers (C-reactive protein [CRP], interleukin-6 [IL-6], procalcitonin [PCT]) were recorded daily. Exploratory plasma proteomics were performed longitudinally in 52 patients using a multiplex proximity extension assay (Olink proteomics). Compared with the CRSonly cohort, we noted increased event-day IL-6 (median 2243 versus 64 pg/mL, P = 0.03) and particularly high PCT levels (median 1.6 versus 0.3 µg/L, P < 0.0001) in the patients that developed severe infections. For PCT, an optimal discriminatory threshold of 1.5 µg/L was established (area under the receiver operating characteristic curve [AUCROC] = 0.78). Next, we incorporated day-of-fever PCT levels with the patient-individual CAR-HEMATOTOX score. In a multicenter validation cohort (n = 125), we confirmed the discriminatory capacity of this so-called HT10 score for early infections at first fever (AUCROC = 0.87, P < 0.0001, sens. 86%, spec. 86%). Additionally, Olink proteomics revealed pronounced immune dysregulation and endothelial dysfunction in patients with severe infections as evidenced by an increased ANGPT2/1 ratio and an altered CD40/CD40L-axis. In conclusion, the high discriminatory capacity of the HT10 score for infections highlights the advantage of dynamic risk assessment and supports the incorporation of PCT into routine inflammatory panels. Candidate markers from Olink proteomics may further refine risk-stratification. If validated prospectively, the score will enable risk-adapted decisions on antibiotic use.

Endothelial deletion of the cytochrome P450 reductase leads to cardiac remodelling.

The cytochrome P450 reductase (POR) transfers electrons to all microsomal cytochrome P450 enzymes (CYP450) thereby driving their activity. In the vascular system, the POR/CYP450 system has been linked to the production of epoxyeicosatrienoic acids (EETs) but also to the generation of reactive oxygen species. In cardiac myocytes (CMs), EETs have been shown to modulate the cardiac function and have cardioprotective effects. The functional importance of the endothelial POR/CYP450 system in the heart is unclear and was studied here using endothelial cell-specific, inducible knockout mice of POR (ecPOR-/-). RNA sequencing of murine cardiac cells revealed a cell type-specific expression of different CYP450 homologues. Cardiac endothelial cells mainly expressed members of the CYP2 family which produces EETs, and of the CYP4 family that generates omega fatty acids. Tamoxifen-induced endothelial deletion of POR in mice led to cardiac remodelling under basal conditions, as shown by an increase in heart weight to body weight ratio and an increased CM area as compared to control animals. Endothelial deletion of POR was associated with a significant increase in endothelial genes linked to protein synthesis with no changes in genes of the oxidative stress response. CM of ecPOR-/- mice exhibited attenuated expression of genes linked to mitochondrial function and an increase in genes related to cardiac myocyte contractility. In a model of pressure overload (transverse aortic constriction, TAC with O-rings), ecPOR-/- mice exhibited an accelerated reduction in cardiac output (CO) and stroke volume (SV) as compared to control mice. These results suggest that loss of endothelial POR along with a reduction in EETs leads to an increase in vascular stiffness and loss in cardioprotection, resulting in cardiac remodelling.

Epoxyeicosatrienoic Acid and Prostanoid Crosstalk at the Receptor and Intracellular Signaling Levels to Maintain Vascular Tone.

Epoxyeicosatrienoic acids (EETs) are signaling lipids produced by the cytochrome P450-(CYP450)-mediated epoxygenation of arachidonic acid. EETs have numerous biological effects on the vascular system, but aspects including their species specificity make their effects on vascular tone controversial. CYP450 enzymes require the 450-reductase (POR) for their activity. We set out to determine the contribution of endothelial CYP450 to murine vascular function using isolated aortic ring preparations from tamoxifen-inducible endothelial cell-specific POR knockout mice (ecPOR-/-). Constrictor responses to phenylephrine were similar between control (CTR) and ecPOR-/- mice. Contrastingly, sensitivity to the thromboxane receptor agonist U46619 and prostaglandin E2 (PGE2) was increased following the deletion of POR. Ex vivo incubation with a non-hydrolyzable EET (14,15-EE-8(Z)-E, EEZE) reversed the increased sensitivity to U46619 to the levels of CTR. EETs had no effect on vascular tone in phenylephrine-preconstricted vessels, but dilated vessels contracted with U46619 or PGE2. As U46619 acts through RhoA-dependent kinase, this system was analyzed. The deletion of POR affected the expression of genes in this pathway and the inhibition of Rho-GTPase with SAR407899 decreased sensitivity to U46619. These data suggest that EET and prostanoid crosstalk at the receptor level and that lack of EET production sensitizes vessels to vasoconstriction via the induction of the Rho kinase system.

Loss of Endothelial Cytochrome P450 Reductase Induces Vascular Dysfunction in Mice.

BACKGROUND: POR (cytochrome P450 reductase) provides electrons for the catalytic activity of the CYP (cytochrome P450) monooxygenases. CYPs are dual-function enzymes as they generate protective vasoactive mediators derived from polyunsaturated fatty acids but also reactive oxygen species. It is not known in which conditions the endothelial POR/CYP system is beneficial versus deleterious. Here, the activity of all CYP enzymes was eliminated in the vascular endothelium to examine its impact on vascular function. METHODS: An endothelial-specific, tamoxifen-inducible POR knockout mouse (ecPOR-/-) was generated. Vascular function was studied by organ chamber experiments. eNOS (endothelial nitric oxide synthase) activity was accessed by heavy arginine/citrulline LC-MS/MS detection and phosphorylation of serine1177 in aortic rings. CYP-derived epoxyeicosatrienoic acids and prostanoids were measured by LC-MS/MS. Gene expression of aorta and endothelial cells was profiled by RNA sequencing. Blood pressure was measured by telemetry. RESULTS: Acetylcholine-induced endothelium-dependent relaxation was attenuated in isolated vessels of ecPOR-/- as compared with control mice. Additionally, ecPOR-/- mice had attenuated eNOS activity and eNOS/AKT phosphorylation. POR deletion reduced endothelial stores of CYP-derived epoxyeicosatrienoic acids but increased vascular prostanoids. This phenomenon was paralleled by the induction of genes implicated in eicosanoid generation. In response to Ang II (angiotensin II) infusion, blood pressure increased significantly more in ecPOR-/- mice. Importantly, the cyclooxygenase inhibitor Naproxen selectively lowered the Ang II-induced hypertension in ecPOR-/- mice. CONCLUSIONS: POR expression in endothelial cells maintains eNOS activity and its loss results in an overactivation of the vasoconstrictor prostanoid system. Through these mechanisms, loss of endothelial POR induces vascular dysfunction and hypertension.

Reactive Oxygen Species Differentially Modulate the Metabolic and Transcriptomic Response of Endothelial Cells.

Reactive oxygen species (ROS) are important mediators of both physiological and pathophysiological signal transduction in the cardiovascular system. The effects of ROS on cellular processes depend on the concentration, localization, and duration of exposure. Cellular stress response mechanisms have evolved to mitigate the negative effects of acute oxidative stress. In this study, we investigate the short-term and long-term metabolic and transcriptomic response of human umbilical vein endothelial cells (HUVEC) to different types and concentrations of ROS. To generate intracellular H2O2, we utilized a lentiviral chemogenetic approach for overexpression of human D-amino acid oxidase (DAO). DAO converts D-amino acids into their corresponding imino acids and H2O2. HUVEC stably overexpressing DAO (DAO-HUVEC) were exposed to D-alanine (3 mM), exogenous H2O2 (10 µM or 300 µM), or menadione (5 µM) for various timepoints and subjected to global untargeted metabolomics (LC-MS/MS) and RNAseq by MACE (Massive analysis of cDNA ends). A total of 300 µM H2O2 led to pronounced changes on both the metabolic and transcriptomic level. In particular, metabolites linked to redox homeostasis, energy-generating pathways, and nucleotide metabolism were significantly altered. Furthermore, 300 µM H2O2 affected genes related to the p53 pathway and cell cycle. In comparison, the effects of menadione and DAO-derived H2O2 mainly occurred at gene expression level. Collectively, all types of ROS led to subtle changes in the expression of ribosomal genes. Our results show that different types and concentration of ROS lead to a different metabolic and transcriptomic response in endothelial cells.

Broadband mid-infrared phase retrieval for nonlinear microscopy.

Spectral phase characterization of ultrashort laser pulses is essential in nonlinear micro-spectroscopy. Whereas in many applications phases are determined for near-infrared (NIR) pulses, successful mid-infrared (MIR) phase retrieval is rare. The spectral phase of ultra-broadband MIR pulses is determined over more than 1000cm-1 in the presented work. This is accomplished by exploiting the d-scan method in two variants. Both allow for detecting high signals by using the interaction of the MIR and NIR pulses. The two variants differ in imprinting the dispersion. While the dual d-scan imprints phases on both pulses, the Xd-scan method disperses the NIR pulses solely.

Nox4 Maintains Blood Pressure during Low Sodium Diet.

The NADPH oxidase Nox4 is a hydrogen peroxide (H2O2)-producing enzyme, with the highest expression in the kidney. As the kidney is involved in volume and blood pressure control through sodium handling, we set out to determine the impact of a low sodium diet on these parameters in WT and Nox4-/- mice. Nox4 expression in the murine kidney was restricted to the proximal tubule. Nevertheless, low-sodium-induced weight loss and sodium sparing function was similar in WT and Nox4-/- mice, disputing an important function of renal Nox4 in sodium handling. In contrast, a low sodium diet resulted in a reduction in systolic blood pressure in Nox4-/- as compared to WT mice. This was associated with a selectively lower pressure to heart-rate ratio, as well as heart to body weight ratio. In general, a low sodium diet leads to activation of sympathetic tone and the renin angiotensin system, which subsequently increases peripheral resistance. Our observations suggest that the control by this system is attenuated in Nox4-/- mice, resulting in lower blood pressure in response to low sodium.

Acousto-optic modulator based dispersion scan for phase characterization and shaping of femtosecond mid-infrared pulses.

Compression, shaping and characterization of broadband mid-infrared (MIR) pulses based on an acousto-optic modulator (AOM) pulse shaper is presented. Characterization of the spectral phase is achieved by an AOM-shaper based implementation of a dispersion scan (d-scan). The abilities of the setup are demonstrated by imprinting several test phases with increasing complexity on broadband MIR pulses centered at 3.2 µm and retrieval of the imprinted phases with the presented d-scan method. Phase characterization with d-scan in combination with an evolutionary algorithm allows us to compress the MIR pulses below 50 fs FWHM autocorrelation after the shaper.

Oxidation of HDAC4 by Nox4-derived H2O2 maintains tube formation by endothelial cells.

NADPH oxidases produce reactive oxygen species that differ in localization, type and concentration. Within the Nox family only Nox4 produces H2O2 which can directly oxidize cysteine residues. With this post-translational modification, activity, stability, localization and protein-protein interactions of the affected protein is altered. Nox4 controls differentiation, cellular homeostasis and prevents inflammation. Therefore, is likely that epigenetic mechanisms contribute to the effects of Nox4. One group of epigenetic modifiers are class IIa histone deacetylases (HDACs). We hypothesize that Nox4-derived H2O2 oxidizes HDACs and analyzed whether HDACs can be differentially oxidized by Nox4. As an artificial system, we utilized HEK293 cells, overexpressing Nox4 in a tetracycline-inducible manner. HDAC4 was oxidized upon Nox4 overexpression. Additionally, Nox4 overexpression increased HDAC4 phosphorylation on Ser632. H2O2 disrupted HDAC4/Mef2A complex, which de-represses Mef2A. In endothelial cells such as HUVECs and HMECs, overexpression of HDAC4 significantly reduced tube formation. Overexpression of a redox insensitive HDAC4 had no effect on endothelial tube formation. Treatment with H2O2, induction of Nox4 expression by treatment of the cells with TGFß and co-overexpression of Nox4 not only induced phosphorylation of HDAC4, but also restored the repressive effect of HDAC4 for tube formation, while overexpression of a redox dead mutant of Nox4 did not. Taken together, Nox4 oxidizes HDAC4, increases its phosphorylation, and eventually ensures proper tube formation by endothelial cells.

NADPH oxidase subunit NOXO1 is a target for emphysema treatment in COPD.

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and death worldwide. Peroxynitrite, formed from nitric oxide, which is derived from inducible nitric oxide synthase, and superoxide, has been implicated in the development of emphysema, but the source of the superoxide was hitherto not characterized. Here, we identify the non-phagocytic NADPH oxidase organizer 1 (NOXO1) as the superoxide source and an essential driver of smoke-induced emphysema and pulmonary hypertension development in mice. NOXO1 is consistently upregulated in two models of lung emphysema, Cybb (also known as NADPH oxidase 2, Nox2)-knockout mice and wild-type mice with tobacco-smoke-induced emphysema, and in human COPD. Noxo1-knockout mice are protected against tobacco-smoke-induced pulmonary hypertension and emphysema. Quantification of superoxide, nitrotyrosine and multiple NOXO1-dependent signalling pathways confirm that peroxynitrite formation from nitric oxide and superoxide is a driver of lung emphysema. Our results suggest that NOXO1 may have potential as a therapeutic target in emphysema.

Author Correction: NADPH oxidase subunit NOXO1 is a target for emphysema treatment in COPD.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In-Ear Pulse Rate Measurement: A Valid Alternative to Heart Rate Derived from Electrocardiography?

Heart rate measurement has become one of the most widely used methods of monitoring the intensity of physical activity. The purpose of this study was to assess whether in-ear photoplethysmographic (PPG) pulse rate (PR) measurement devices represent a valid alternative to heart rate derived from electrocardiography (ECG), which is considered a gold standard. Twenty subjects (6 women, 14 men) completed one trial of graded cycling under laboratory conditions. In the trial, PR was recorded by two commercially available in-ear devices, the Dash Pro and the Cosinuss°One. They were compared to HR measured by a Bodyguard2 ECG. Validity of the in-ear PR measurement devices was tested by ANOVA, mean absolute percentage errors (MAPE), intra-class correlation coefficient (ICC), and Bland-Altman plots. Both devices achieved a MAPE ≤5%. Despite excellent to good levels of agreement, Bland-Altman plots showed that both in-ear devices tend to slightly underestimate the ECG's HR values. It may be concluded that in-ear PPG PR measurement is a promising technique that shows accurate but imprecise results under controlled conditions. However, PPG PR measurement in the ear is sensitive to motion artefacts. Thus, accuracy and precision of the measured PR depend highly on measurement site, stress situation, and exercise.

Towards a quantum cascade laser-based implant for the continuous monitoring of glucose.

Continuous glucose monitoring enables an improved disease management for people with diabetes. However, state-of-the-art, enzyme-based, minimally invasive sensors lose their sensitivity over time and have to be replaced periodically. Here, we present the in vitro investigation of a quantum cascade laser-based measurement scheme that conceptually should be applicable over elongated periods of time due to its reagent-free nature and may therefore be considered as an approach towards long-term implantation. The method uses a miniaturized optofluidic interface in transflection geometry to measure the characteristic mid-infrared absorption properties of glucose. A glucose sensitivity of 3.2 mg dL-1 is achieved in aqueous glucose solutions. While this sensitivity drops to 12 mg dL-1 in the presence of biologically plausible, maximum concentrations of other monosaccharides, it is still well within the medically acceptable range according to Parkes error grid analysis. With a response time of less than five minutes, our sensor should be able to react adequately fast to physiological changes in glucose concentration. Finally, no drift or deterioration was found during an extended, 42 days in vitro experiment. These results underline the potential of this technique for its conceivable applicability in vivo as a long-term glucose monitoring implant.

Flexible and broadly tunable infrared light source based on shaped sub-10-fs pulses for a multimodal microscopy setup.

We present a versatile approach for mid-infrared spectroscopy through the flexible control of a difference-frequency-generation (DFG) process by femtosecond (fs) pulse shaping and spectral focusing. Based on a broadband sub-10-fs oscillator, the spectral position and spectral resolution can be independently selected within the molecular fingerprint region of more than 2000 cm-1. A spectral resolution better than 20 cm-1 can be achieved, which depends solely on the pulse shaper configuration. An absorption experiment on a polystyrene reference sample finally validates the concept and opens the door for an additional modality in nonlinear multimodal microscopy setups.

Efficacy and Safety of Mycophenolate Mofetil and Tacrolimus as Second-line Therapy for Patients With Autoimmune Hepatitis.

BACKGROUND & AIMS: Predniso(lo)ne, alone or in combination with azathioprine, is the standard-of-care (SOC) therapy for autoimmune hepatitis (AIH). However, the SOC therapy is poorly tolerated or does not control disease activity in up to 20% of patients. We assessed the efficacy of mycophenolate mofetil (MMF) and tacrolimus as second-line therapy for patients with AIH. METHODS: We performed a retrospective study of data (from 19 centers in Europe, the United States, Canada, and China) from 201 patients with AIH who received second-line therapy (121 received MMF and 80 received tacrolimus), for a median of 62 months (range, 6-190 mo). Patients were categorized according to their response to SOC. Patients in group 1 (n = 108) had a complete response to the SOC, but were switched to second-line therapy as a result of side effects of predniso(lo)ne or azathioprine, whereas patients in group 2 (n = 93) had not responded to SOC. RESULTS: There was no significant difference in the proportion of patients with a complete response to MMF (69.4%) vs tacrolimus (72.5%) (P = .639). In group 1, MMF and tacrolimus maintained a biochemical remission in 91.9% and 94.1% of patients, respectively (P = .682). Significantly more group 2 patients given tacrolimus compared with MMF had a complete response (56.5% vs 34%, respectively; P = .029) There were similar proportions of liver-related deaths or liver transplantation among patients given MMF (13.2%) vs tacrolimus (10.3%) (log-rank, P = .472). Ten patients receiving MMF (8.3%) and 10 patients receiving tacrolimus (12.5%) developed side effects that required therapy withdrawal. CONCLUSIONS: Long-term therapy with MMF or tacrolimus generally was well tolerated by patients with AIH. The agents were equally effective in previous complete responders who did not tolerate SOC therapy. Tacrolimus led to a complete response in a greater proportion of previous nonresponder patients compared with MMF.

Chiral Magnetic Effect and Anomalous Transport from Real-Time Lattice Simulations.

We present a first-principles study of anomaly induced transport phenomena by performing real-time lattice simulations with dynamical fermions coupled simultaneously to non-Abelian SU(N_{c}) and Abelian U(1) gauge fields. Investigating the behavior of vector and axial currents during a sphaleron transition in the presence of an external magnetic field, we demonstrate how the interplay of the chiral magnetic and chiral separation effect leads to the formation of a propagating wave. We further analyze the dependence of the magnitude of the induced vector current and the propagation of the wave on the amount of explicit chiral symmetry breaking due to finite quark masses.

Infection Risk in Sterile Operative Procedures.

BACKGROUND: The main objective of hospital hygiene and infection prevention is to protect patients from preventable nosocomial infections. It was recently stated that the proper goal should be for zero infection rates in sterile surgical procedures. In this article, we attempt to determine whether this demand is supported by the available literature. METHODS: We systematically searched the Medline and EMBASE databases for studies published in the last 10 years on the efficacy of infection control measures and carried out a meta-analysis according to the PRISMA tool. We used the following search terms: "aseptic surgery," "intervention," "surgical site infection," "nosocomial infection," "intervention," and "prevention." RESULTS: 2277 articles were retrieved, of which 204 were acquired in full text and analyzed. The quantitative analysis included 7 prospective cohort studies on the reduction of nosocomial infection rates after aseptic surgery. The measures used included training sessions, antibiotic prophylaxis, and operative-site disinfection and cleaning techniques. These interventions succeeded in reducing postoperative wound infections (relative risk (RR] 0.99 [0.98; 1.00]). Subgroup analyses on antibiotic prophylaxis (RR 0.99 [0.98; 1.01]) and noncontrolled trials (RR 0.97 [0.92; 1.02]) revealed small, insignificant effects. CONCLUSION: A multimodal approach with the participation of specialists from various disciplines can further reduce the rate of postoperative infection. A reduction to zero is not realistic and is not supported by available evidence.

Case report: severe cytomegalovirus primary infection in an immunocompetent adult with disseminated intravascular coagulation treated with valganciclovir.

BACKGROUND: Disseminated intravascular coagulation (DIC) is a very rare complication of disseminated cytomegalovirus (CMV) infection. So far it is mainly described for immunocompromised patients. CASE PRESENTATION: A 49-year-old immunocompetent Caucasian male presented with sudden onset of fever and DIC due to primary CMV infection, which was treated with Valganciclovir. CMV-specific IgG-avidity and epithelial cell-specific neutralisation-capacity developed five weeks after onset of symptoms. We describe the first case of an immunocompetent patient suffering from DIC due to a CMV primary infection successfully treated with Valganciclovir. CONCLUSIONS: Primary CMV infection can occur accompanied with life threatening complications even in immunocompetent patients. Immediate treatment with Valganciclovir should be considered as an early treatment of choice in severe cases since specific neutralisation capacity might need several weeks to develop.

Temporal resolution beyond the average pulse duration in shaped noisy-pulse transient absorption spectroscopy.

In time-resolved spectroscopy, it is a widespread belief that the temporal resolution is determined by the laser pulse duration. Recently, it was observed and shown that partially coherent laser pulses as they are provided by free-electron-laser (FEL) sources offer an alternative route to reach a temporal resolution below the average pulse duration. Here, we demonstrate the generation of partially coherent light in the laboratory like we observe it at FELs. We present the successful implementation of such statistically fluctuating pulses by using the pulse-shaping technique. These pulses exhibit an average pulse duration about 10 times larger than their bandwidth limit. The shaped pulses are then applied to transient-absorption measurements in the dye IR144. Despite the noisy characteristics of the laser pulses, features in the measured absorption spectra occurring on time scales much faster than the average pulse duration are resolved, thus proving the universality of the described noisy-pulse concept.

Signatures and control of strong-field dynamics in a complex system.

Controlling chemical reactions by light, i.e., the selective making and breaking of chemical bonds in a desired way with strong-field lasers, is a long-held dream in science. An essential step toward achieving this goal is to understand the interactions of atomic and molecular systems with intense laser light. The main focus of experiments that were performed thus far was on quantum-state population changes. Phase-shaped laser pulses were used to control the population of final states, also, by making use of quantum interference of different pathways. However, the quantum-mechanical phase of these final states, governing the system's response and thus the subsequent temporal evolution and dynamics of the system, was not systematically analyzed. Here, we demonstrate a generalized phase-control concept for complex systems in the liquid phase. In this scheme, the intensity of a control laser pulse acts as a control knob to manipulate the quantum-mechanical phase evolution of excited states. This control manifests itself in the phase of the molecule's dipole response accessible via its absorption spectrum. As reported here, the shape of a broad molecular absorption band is significantly modified for laser pulse intensities ranging from the weak perturbative to the strong-field regime. This generalized phase-control concept provides a powerful tool to interpret and understand the strong-field dynamics and control of large molecules in external pulsed laser fields.